阅读说明：本技术 人源化抗Aβ单克隆抗体及其应用 (Humanized anti-Abeta monoclonal antibody and application thereof ) 是由 冯晓 王涛 金磊 车翀 梁阳秋 刘爽 孙丹丹 王英武 秦锁富 滕国生 于 2019-02-01 设计创作，主要内容包括：本发明涉及抗体药物技术领域,尤其涉及人源化抗Aβ单克隆抗体及其应用。本发明提供的人源化抗Aβ单克隆抗体能够抑制Aβ单体聚合,促进巨噬细胞对Aβ的吞噬,保护神经细胞免受Aβ的毒性,可用于治疗和诊断淀粉样变性相关的疾病和病症,如阿尔茨海默病。(The invention relates to the technical field of antibody medicines, in particular to a humanized anti-Abeta monoclonal antibody and application thereof. The humanized anti-A beta monoclonal antibody provided by the invention can inhibit polymerization of an A beta monomer, promote phagocytosis of the A beta by macrophages, protect nerve cells from toxicity of the A beta, and can be used for treating and diagnosing diseases and symptoms related to amyloidosis, such as Alzheimer's disease.)

1. A humanized anti-A beta monoclonal antibody characterized in that,

(I) the amino acid sequences of the three CDR regions of the heavy chain of the monoclonal antibody respectively have the amino acid sequences shown as SEQ ID NO 1, 2 and 3; and (II) the amino acid sequences of the three CDR regions of the light chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO 4, 5 and 6 respectively;

or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same function with the amino acid sequence of (I) or (II);

or

(IV) an amino acid sequence having a homology of 97% or more with the amino acid sequence of (I), (II) or (III).

2. The monoclonal antibody of claim 1,

(V) the amino acid sequences of the 4 FR regions of the heavy chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO:7, 8, 9 and 10, respectively; and (VI), the amino acid sequences of the 4 FR regions of the light chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO. 11, 12, 13 and 14 respectively;

or

(VII), (V) or (VI) by substituting, deleting or adding one or more amino acids, and is functionally identical to the amino acid sequence (V) or (VI);

or

(VIII) an amino acid sequence having a homology of 97% or more with the amino acid sequence of (V), (VI) or (VII).

3. The monoclonal antibody of claim 1,

(IX) the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NO 15-19, and (X) the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 20-24;

or

(XI), (IX) or (X) by substitution, deletion or addition of one or more amino acids, and is functionally identical to the amino acid sequence of (IX) or (X);

or

(XII) or an amino acid sequence having a homology of 97% or more with the amino acid sequence described In (IX), (X) or (XI).

4. The monoclonal antibody of claim 1 or 3, wherein the epitopes of the monoclonal antibody are both A β_14～29。

5. The monoclonal antibody of claim 1 or 3,

(XIII) in which the heavy chain variable region has an amino acid sequence shown in SEQ ID NO:15 and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 23;

or (XIV) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24;

or (XV) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20;

or (XVI) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 21;

or (XVII) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 22;

or (XVIII) has the amino acid sequence shown in SEQ ID NO:19 in its heavy chain variable region and the amino acid sequence shown in SEQ ID NO:23 in its light chain variable region.

6. The monoclonal antibody of any one of claims 1 to 5, wherein the plurality is 2, 3, 4 or 5.

7. The monoclonal antibody of any one of claims 1-6, further comprising a constant region, the constant region of the heavy chain of the monoclonal antibody being any one of human IgG1, IgG2, IgG3, or IgG 4; the constant region of the light chain of the monoclonal antibody is of the kappa type or of the lambda type.

8. A nucleotide encoding the monoclonal antibody of any one of claims 1 to 7.

9. An expression vector comprising nucleotides encoding the monoclonal antibody of any one of claims 1 to 7.

10. A host cell transformed or transfected with the expression vector of claim 9.

11. A method of producing a monoclonal antibody as claimed in any one of claims 1 to 7, comprising: culturing the host cell of claim 10, and inducing expression of the humanized anti-a β monoclonal antibody.

12. A conjugate comprising a chemically or biologically labeled monoclonal antibody according to any one of claims 1-7.

13. A monoclonal antibody according to any one of claims 1 to 7 or a conjugate according to claim 12 conjugated to a solid or semi-solid medium.

14. Use of the monoclonal antibody of any one of claims 1 to 7, the conjugate of claim 12 and/or the conjugate of claim 13 for the preparation of an agent against cognitive impairment, a therapeutic agent for alzheimer's disease, an agent for suppressing the progression of alzheimer's disease, an agent for inhibiting the formation of age spots, an agent for suppressing the accumulation of a β, an anti-neurotoxic agent, an agent for inhibiting the formation of a β amyloid fibrils and/or an agent against synaptic toxicity.

15. Use of a monoclonal antibody according to any one of claims 1 to 7, a conjugate according to claim 12 and/or a conjugate according to claim 13 for the manufacture of a medicament for the prevention or treatment of a disease;

the disease includes amyloidosis, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

16. Medicament comprising a monoclonal antibody according to any one of claims 1 to 7, a conjugate according to claim 12 and/or a conjugate according to claim 13.

17. A method for the prophylaxis and/or treatment of a disease, characterized by administering a medicament according to claim 16; the disease includes amyloidosis, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

18. Use of a monoclonal antibody according to any one of claims 1 to 7, a conjugate according to claim 12 and/or a conjugate according to claim 13 for the preparation of a product for detecting expression of a β.

19. A kit comprising a monoclonal antibody according to any one of claims 1 to 7, a conjugate according to claim 12 and/or a conjugate according to claim 13.

20. A method for diagnosing a disease, comprising detecting a β expression using the kit according to claim 19, and determining whether or not a disease is present based on the amount of a β expression;

the disease includes amyloidosis, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a humanized anti-Abeta monoclonal antibody and application thereof.

Background

Aβ

Amyloid beta (a β) is encoded by the human chromosome 21 gene, contains 39-43 amino acids, is in a β -sheet structure, is hydrophobic, and has a molecular weight of 4 KDa. A beta is derived from residue polypeptide generated by proteolytic enzyme cleavage of Amyloid Precursor Protein (APP). APP can be decomposed by alpha, beta and gamma-protease, and the decomposed products have different biological functions. Wherein the sequential action of the beta-protease and the gamma-protease produces A beta. Gamma-proteases generate the C-terminus of a β, which cleaves at the APP transmembrane region and can then generate a large number of 39-43 amino acid residue isoforms. The most common of all residue subtypes are a β 40 and a β 42. The former is typically formed by cleavage of APP at the endoplasmic reticulum, while the latter is formed across the Golgi network.

Pathological mechanism of A β

It is now known that all nerve cells in the Central Nervous System (CNS), including neurons, astrocytes, microglia and endothelial cells, express APP and produce a β. Under normal physiological conditions, APP is hydrolyzed by α -secretase to produce a soluble sAPP α fragment. This fragment contains the extracellular region of APP and the 83 amino acid C-terminus located on the envelope. sAPP α can regulate neuronal excitability, improve synaptic plasticity, learning and memory, and enhance neuronal resistance to oxidative and metabolic stress. Under neuropathological conditions, APP is first hydrolyzed by β -secretase 1(BACE) to produce a fragment of sAPP β and a cell membrane-bound 99 amino acid peptide (C99). Subsequently, the C99 peptide fragment was acted upon by γ -secretase to produce a β. Unlike sappa α, a β causes loss of neurosynaptic function, decreases neuronal plasticity, changes in cellular energy metabolism, induces oxidative stress and mitochondrial dysfunction, thereby causing intracellular calcium imbalance. The formation, aggregation and deposition of a β, and in particular a β 42, can trigger neurotoxic and neurodegenerative diseases, and is also an important ring in the pathogenesis of Alzheimer's Disease (AD).

A β and Alzheimer's Disease (AD)

A beta is one of main pathological marker proteins in the brain of Alzheimer's disease, and the formation, deposition and degradation of the A beta run through the whole pathological process of AD. The A beta is divided into soluble A beta and insoluble A beta, the soluble A beta is not neurotoxic, the soluble A beta has a neurocytotoxicity effect after the beta-folding to form filamentous fiber aggregates which are changed into insoluble precipitates, and the primary structure of the human A beta is a determining factor of the neurocytotoxicity effect. According to the literature, the neurotoxicity of a β is mainly reflected in the following 4 aspects: cholinergic neuronal damage, neuronal apoptosis, peroxidative damage and inflammatory responses.

1. Cholinergic neuronal injury

Massive damage and loss of neurons and synapses of the cholinergic system of the anterior basal zone, which project into the hippocampus and cortex, are the major causes of memory and cognitive decline in AD patients. The A beta activates protein kinase GSK-3/glycogen synthase kinase-3 beta, tau protein and mitochondrial pyruvate dehydrogenase are phosphorylated, the enzyme activity is reduced, and the conversion of pyruvate into acetyl coenzyme A (acyl coenzyme A) is reduced, so that the synthesis of acetylcholine (ACh) is reduced, the succinate dehydrogenase is inhibited, the energy supply is reduced, the damage, degeneration and transmitter transmission disorder of cholinergic neurons and synapses are caused, and the activity of the cholinergic system is reduced. The decrease in ACh, in turn, leads to increased production of a β, which in turn, leads to the formation of a vicious circle.

2. Apoptosis of nerve cells

The main hallmark of AD is a reduction in the number of neurons in the cortex and hippocampus. When A beta is gathered into a beta-sheet layer folding structure, the neurotoxicity is obviously enhanced, and the nerve cell apoptosis can be induced. This is an important cause of selective neuronal and synaptic deficits in AD. The fibrillar aggregated A beta interacts and cross-links with transmembrane receptors such as APP on the cell surface through a secretion pathway, so that the inhibition and abnormal activation of a signal transduction pathway are caused, and a nerve cell death program is started. The unbalance of Ca2+ in the internal environment caused by A beta stimulates an NMDA receptor or causes the change of membrane permeability through the damage effect of free radicals, and the inflow of Ca2+ causes the activation of a glutamate receptor, so that glutamatergic neurons are excited excessively and die. In addition, a β can also cause increased NO synthesis leading to neuronal apoptosis.

3. Damage by peroxidation

A β can cause oxidative stress in a variety of ways. The induction of increased free radicals by a β is an important cause of oxidative stress. Toxicity of a β is mediated by H2O2, and a β increases accumulation of H2O2 in vivo via the Receptor of Advanced Glycation Endproducts (RAGE), causing oxidative damage and cell death. In addition, oxygen stress proliferates microglia, migrating along a β concentration gradient, causing microglia around senile plaques to aggregate, forming neuritic plaques, producing more reactive oxygen radicals. The A beta can also increase lipid peroxidation, and H2O2 is not only a source of hydroxyl free radical, but also increases the abnormal expression of fast response transcription Nuclear Factor kappa B (NF-kappa B) protein, generates nerve cell membrane damage and causes the degeneration of neurons.

4. Inflammatory reaction

There is an inflammatory response in the brain of AD patients. Glia cell hyperplasia around plaques and neurofibrillary tangles, a β stimulates the release of a range of strongly neurotoxic inflammatory proteins, such as a β -activated astrocytes and microglia, to release inflammatory cytokines. Such as NO, interleukin-1 (IL-1, IL-1 can make cytoskeletal protein-neurofilament protein abnormal production, damage neuron function), interleukin-6 (IL-6, IL-6 increase the over-high gene expression of ADP, promote the formation of A beta, the A beta can induce the expression of microglia IL-6, form the vicious circle in AD immune pathological process), tumor necrosis factor-alpha (TNF-alpha, TNF-alpha participates in AD pathological process through the CNS most main apolipoprotein ApoE), gamma-interferon (gamma-IFN), beta-Antitrypsin (ACT), complement C1, C3 and chemotactic factor, adhesion factor, etc. Many inflammatory factors induce inflammatory reactions, promote free radical production, oxidative stress, and cause degeneration and necrosis of nerve cells.

Medicine aiming at A β target point intervention and treatment mechanism

Based on the above, toxicity of a β to neurons is an important factor in the development of AD. Thus, by inhibiting a β production and accelerating its clearance, it will be possible to halt the AD disease process and alleviate the disease symptoms. Drugs currently being developed and used in clinical applications are also based on the mechanism of production and clearance of a β, and include the following components:

1. inhibition of beta-and gamma-secretase

Beta-secretase hydrolysis of APP is the initial stage of amyloid production. Inhibition of β -secretase activity, while inhibiting a β production, can cause major side effects. Since, in addition to APP, β -secretase has numerous substrates, the hydrolysis of which plays an important role in the plasticity of neurons and synapses within the nervous system. Clinically, inhibitors of β -secretase, such as E2609(clinical trial ID # NCT01600859), MK-8931(NCT01739348) and LY2886721(NCT01807026, NCT01561430) are able to reduce a β levels in human cerebrospinal fluid by 80-90%, but no β -secretase inhibitors are currently on the market.

Gamma-secretase hydrolysis of APP is the last step in amyloid production, directly producing a β 40 and a β 42 fragments. Thus, inhibition of γ -secretase is also believed to be effective in inhibiting a β production for the treatment of AD. However, in addition to hydrolyzing APP, γ -secretase also hydrolyzes other substrate proteins, including Notch proteins. Notch proteins are important for cell proliferation, differentiation and intercellular signaling. Semagacestat (LY450139) was used as a gamma-secretase inhibitor in 3000 clinical trials (NCT00762411, NCT01035138, NCT 00762411). The results of the tests show that subjects experience not but no improvement but rather a worsening of cognition and are associated with side effects of weight loss, increased probability of skin cancer and high risk of infection. Other inhibitors of gamma-secretase, such as Avagacestat, also failed clinical trials (NCT00810147, NCT00890890, NCT00810147, NCT 01079819). Selective gamma-secretase modulators (SGSMs) can in theory avoid the side effects of total inhibition of gamma-secretase, inhibiting only the hydrolytic pathway of APP without interfering with other signaling pathways, such as the hydrolysis of Notch proteins. Some nonsteroidal anti-inflammatory drugs, such as ibuprofen, sulindac, indomethacin, and flurbiprofen, can modulate gamma-secretase levels and lower a β 42 levels in vitro and in vivo activity assays. Although this class of drugs has been shown to alleviate mild cognitive impairment and to reduce the levels of inflammatory factors in cerebrospinal fluid, the long-term use of non-steroidal anti-inflammatory drugs for the treatment of AD remains clinically proven.

2. Inhibiting the aggregation of A beta

Inhibition of the formation of age spots can be achieved by interfering with or antagonizing the aggregation of a β. Such as 3-amino-1-propanesulfonic acid (3-APS, alzheimer's, ramiprosate), inhibit a β aggregation by interfering with the interaction of tolerable a β and endogenous aminoglucans, which promote the formation and precipitation of a β amyloid fibers. However, phase III clinical trial results for 3-APS are not ideal, resulting in trial discontinuation. Other anti-a β aggregation drugs have also failed clinically in stages II and III, including Colostrinin, which inhibits a β aggregation and neutralizes a β neurotoxicity in vitro and improves mouse cognition in vivo, but have not achieved satisfactory results in phase II clinical trials. Scyllo-inositol (ELND005) is an oral drug against A β aggregation, and mouse experiments show that Scyllo-inositol can reduce A β toxicity, but does not achieve the expected effect in phase II clinical trials of patients with mild to moderate AD at 18 months.

3. Promoting A beta deposition and polymer removal

There are three major pathways for Α β deposition and polymer removal: activating the activity of amyloid plaque degrading enzymes; modulate the transport of a β in the brain and peripheral circulation and anti-a β immunotherapy.

A β deposits and polymers can be degraded by a variety of proteolytic enzymes, including plasmin, endothelin converting enzyme, angiotensin converting enzyme, metalloproteinases, and the like. These enzymes are present at low levels in the brain of AD patients, but due to the lack of specificity acting on these enzymes, no such drugs are currently in the clinic.

The transport of a β between the central nervous system and the peripheral circulatory system is regulated by apolipoproteins. Low density lipoprotein receptor-related protein (LRP-1) promotes the influx of A β from the brain into the blood. Receptor for advanced glycation end products (RAGE) may assist a β in crossing the blood brain barrier. The therapeutic mechanism is to reduce amyloid load in the brain by limiting the entry of a β into the peripheral circulation. To date, only inhibitors/modulators of RAGE have entered clinical trials, including PF-0449470052, which failed clinically in phase II, and TTP4000, which also had no reliable data indicating expected results in phase I clinical trials.

Anti-a β antibodies can neutralize a β toxicity and can improve the cognition in transgenic animals, and anti-a β antibodies have become popular for AD treatment. Anti-a β antibodies are primarily directed to early treatment of AD, as well as to mild to moderate AD treatment. This is also related to the pathogenic mechanism of a β, which is difficult to reverse and repair once neurons are injured, so early clearance of a β can be a more effective treatment and relief of AD.

4. A beta target antibody in clinical trial

There are currently 15 drugs of anti-a β antibodies in clinical trials, and in comparison, adacanaumab, ganteneumab and Solanezumab have progressed faster and have entered phase III clinics, and as mechanism-addressed, companies have localized the indications to mild senile dementia.

While there is currently some theoretical knowledge in the field of treating and preventing alzheimer's disease, there remains a need for improved compositions and methods for treating and/or preventing the disease, and a need exists for antibodies and treatments that are capable of targeting a β. Although there are some humanized monoclonal antibodies that are highly advantageous in terms of therapeutic treatment, it is at no time easy to screen for humanized monoclonal antibodies having the desired properties and functions, and there is still a pressing need for such humanized monoclonal antibodies in reality.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a humanized anti-a β monoclonal antibody and its application. And provides a vector of the nucleotide for coding the monoclonal antibody, a host cell and application thereof. The antibody gene variable region sequence can constitute full-length antibody molecule as medicine for clinical treatment and diagnosis of amyloidosis relative diseases and diseases, such as Alzheimer's disease.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides humanized anti-A beta monoclonal antibodies,

(I) the amino acid sequences of the three CDR regions of the heavy chain of the monoclonal antibody respectively have the amino acid sequences shown as SEQ ID NO 1, 2 and 3; and (II) the amino acid sequences of the three CDR regions of the light chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO 4, 5 and 6 respectively;

or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same function with the amino acid sequence of (I) or (II);

further, the functions are two or three of inhibiting A beta polymerization, promoting phagocytosis of A beta by macrophages and protecting activity of cytotoxicity;

or

(IV) an amino acid sequence having a homology of 97% or more with the amino acid sequence of (I), (II) or (III).

Further, the epitope of the monoclonal antibody of the invention is A β_14～29。

In some embodiments of the invention, the invention provides humanized anti-a β monoclonal antibodies:

the heavy chain comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has the amino acid sequence shown as SEQ ID NO. 1, 2 or 3, or has the amino acid sequence with more than 97 percent of homology with the CDR region;

the light chain comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO. 4, 5 or 6, or has more than 97 percent of homology with the amino acid sequence.

In the invention, the amino acid sequences of three CDR regions of the heavy chain of the monoclonal antibody respectively have the amino acid sequences shown as SEQ ID NO. 1, 2 and 3;

the amino acid sequences of the three CDR regions of the light chain of the monoclonal antibody have the amino acid sequences shown in SEQ ID NO 4, 5 and 6 respectively.

Wherein, the sequence shown in SEQ ID NO. 1 is NYNIH;

the sequence shown as SEQ ID NO. 2 is AIYPGNGDTTYNQKVKG;

the sequence shown in SEQ ID NO. 3 is GDWDWFAY;

the sequence shown in SEQ ID NO. 4 is SSSKSLLHSNGITYLY;

the sequence shown in SEQ ID NO. 5 is RMSNLAS;

the sequence shown in SEQ ID NO. 6 is AQMLERPLT.

In some embodiments of the invention, the monoclonal antibodies provided herein,

(V) the amino acid sequences of the 4 FR regions of the heavy chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO:7, 8, 9 and 10, respectively; and (VI), the amino acid sequences of the 4 FR regions of the light chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO. 11, 12, 13 and 14 respectively;

or

(VII), (V) or (VI) by substituting, deleting or adding one or more amino acids, and is functionally identical to the amino acid sequence (V) or (VI);

further, the functions are two or three of inhibiting A beta polymerization, promoting phagocytosis of A beta by macrophages and protecting activity of cytotoxicity;

or

(VIII) an amino acid sequence having a homology of 97% or more with the amino acid sequence of (V), (VI) or (VII).

In some embodiments of the invention, the heavy chain comprises 4 FR regions wherein the amino acid sequence of at least one FR region has the amino acid sequence shown in SEQ ID NO 7, 8, 9 or 10, or an amino acid sequence with more than 97% homology thereto;

the light chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 11, 12, 13 or 14, or has the amino acid sequence with more than 97 percent of homology with the FR region.

In some embodiments of the invention, the amino acid sequences of the 4 FR regions of the heavy chain of the monoclonal antibody have the amino acid sequences shown as SEQ ID NO. 7, 8, 9 or 10, respectively, or amino acid sequences having more than 97% homology thereto;

the amino acid sequences of the 4 FR regions of the light chain of the monoclonal antibody respectively have the amino acid sequences shown in SEQ ID NO. 11, 12, 13 or 14, or have the amino acid sequences with over 97 percent of homology with the amino acid sequences.

Wherein the sequence shown in SEQ ID NO. 7 is QVQLVQSGAEVKKPGASVKVSCKASGYTFT;

the sequence shown as SEQ ID NO. 8 is WVRQAPGQRLEWIG;

the sequence shown as SEQ ID NO. 9 is KATLTADKSASTAYMELSSLRSEDTAVYYCAR;

the sequence shown in SEQ ID NO. 10 is WGQGTLVTVSS;

the sequence shown as SEQ ID NO. 11 is DIVMTQTPLSLPVTPGEPASISC;

the sequence shown as SEQ ID NO. 12 is WYLQKPGQSPRLLIY;

sequence GVPDRFSGSGSGTDFTLRISRVEAEDVGVYYC shown in SEQ ID NO. 13;

the sequence shown as SEQ ID NO. 14 is FGQGTKVDIK;

in some embodiments of the invention, the monoclonal antibody,

(IX) the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NO 15-19, and (X) the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 20-24;

or

(XI), (IX) or (X) by substitution, deletion or addition of one or more amino acids, and is functionally identical to the amino acid sequence of (IX) or (X);

further, the functions are two or three of inhibiting A beta polymerization, promoting phagocytosis of A beta by macrophages and protecting activity of cytotoxicity;

or

(XII) or an amino acid sequence having a homology of 97% or more with the amino acid sequence described In (IX), (X) or (XI).

In some embodiments of the invention, the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID NOs 15-19; the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 20-24.

In some embodiments of the invention, the humanized anti a β monoclonal antibody has:

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15,16,17,18,19 and a light chain variable region having an amino acid sequence of SEQ ID NO. 20;

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15,16,17,18,19 and a light chain variable region having an amino acid sequence of SEQ ID NO. 21;

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15,16,17,18,19 and a light chain variable region having an amino acid sequence of SEQ ID NO. 22;

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15,16,17,18,19 and a light chain variable region having an amino acid sequence of SEQ ID NO. 23;

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15,16,17,18,19 and a light chain variable region having an amino acid sequence of SEQ ID NO. 24;

in some embodiments of the invention, the monoclonal antibody,

(XIII) in which the heavy chain variable region has an amino acid sequence shown in SEQ ID NO:15 and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 23;

or (XIV) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24;

or (XV) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20;

or (XVI) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 21;

or (XVII) the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 22;

or (XVIII) has the amino acid sequence shown in SEQ ID NO:19 in its heavy chain variable region and the amino acid sequence shown in SEQ ID NO:23 in its light chain variable region.

In the present invention, the sequence having at least 97% sequence homology is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids based on the original sequence, and the plurality is 2, 3, 4 or 5.

In some embodiments of the invention, the monoclonal antibody of the invention further comprises a constant region, wherein the constant region of the heavy chain of the monoclonal antibody is any one of human IgG1, IgG2, IgG3 or IgG 4; the constant region of the light chain of the monoclonal antibody is of the kappa type or of the lambda type.

The heavy chain constant region of the humanized anti-Abeta monoclonal antibody provided by the invention is human IgG1, and the light chain constant region is the constant region of a human kappa chain.

The humanized anti-Abeta monoclonal antibody provided by the invention can be combined with human Abeta; in certain embodiments, the affinity between an antibody and its target is characterized by Ka (binding constant), Kd (dissociation constant), Kd (equilibrium dissociation constant), the invention provides that the antibody has a Kd value of no greater than 5.96 nM; the humanized anti-A beta monoclonal antibody provided by the invention can inhibit A beta monomer polymerization, promote phagocytosis of A beta by macrophages and protect nerve cells from the toxicity of A beta.

The invention also provides the nucleotide for coding the monoclonal antibody.

The invention provides a nucleotide sequence for encoding the heavy chain of the monoclonal antibody.

The invention provides a nucleotide sequence for encoding the monoclonal antibody light chain.

The invention provides a nucleotide sequence for encoding the heavy chain variable region of the monoclonal antibody.

Wherein, the nucleotide sequence for coding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 25-29 or is a complementary sequence of SEQ ID NO. 25-29.

In some embodiments of the present invention, the nucleotide has a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in any one of SEQ ID NOs.25 to 29, and has the same or similar function as the nucleotide sequence shown in any one of SEQ ID NOs.25 to 29.

In some embodiments of the invention, the nucleotide sequence shown in any one of SEQ ID NO 25-29 is obtained by substituting, deleting or adding one or more nucleotides, wherein the nucleotides are 2, 3, 4 or 5.

The invention provides a nucleotide sequence for encoding the variable region of the monoclonal antibody light chain.

Wherein the nucleotide sequence for coding the variable region of the monoclonal antibody light chain is shown as SEQ ID NO 30-34 or is a complementary sequence of SEQ ID NO 30-34.

In some embodiments of the present invention, the nucleotide has a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in any one of SEQ ID NOs 30 to 34, and has the same or similar function as the nucleotide sequence shown in any one of SEQ ID NOs 30 to 34.

In some embodiments of the present invention, the nucleotide sequence shown in any one of SEQ ID NO 30-34 is obtained by substituting, deleting or adding one or more nucleotides, wherein the plurality is 2, 3, 4 or 5.

The expression vector provided by the invention comprises nucleotide for coding the anti-Abeta monoclonal antibody.

The invention also provides a host cell transformed or transfected with the expression vector.

The preparation method of the anti-Abeta monoclonal antibody comprises the following steps: culturing the host cell, and inducing the expression of the anti-Abeta monoclonal antibody.

The invention also provides the anti-Abeta monoclonal antibody which is chemically marked or biological marked.

The chemical label is an isotope, an immunotoxin and/or a chemical drug;

the biomarker is a biotin, avidin, or enzyme label.

The enzyme label is preferably horseradish peroxidase or alkaline phosphatase.

The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, modeccin, PAP, nystatin, gelonin or luffa toxin.

The invention relates to a conjugate prepared by coupling an anti-Abeta monoclonal antibody or a conjugate thereof with a solid medium or a semisolid medium.

The solid medium or the non-solid medium is selected from colloidal gold, polystyrene flat plates or beads.

The invention also provides the use of the monoclonal antibody, the conjugate and/or the conjugate in the preparation of an agent against cognitive impairment, a therapeutic agent for alzheimer's disease, an agent for suppressing the progression of alzheimer's disease, an agent for inhibiting the formation of senile plaques, an agent for suppressing the accumulation of a β, an anti-neurotoxic agent, an agent for inhibiting the formation of a β amyloid fibrils and/or an agent against synaptic toxicity.

The invention also provides the application of the humanized anti-Abeta monoclonal antibody, the conjugate and/or the conjugate in the preparation of medicaments for preventing and treating diseases;

the diseases include amyloidosis, which is a disease and abnormality associated with amyloid protein, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

The invention also provides medicaments comprising the humanized anti-a β monoclonal antibodies, conjugates and/or conjugates thereof.

The present invention also provides a method for the prophylaxis and/or treatment of a disease, comprising administering the agent of the present invention; the diseases and disorders include amyloidosis, which is a disease and disorder associated with amyloid protein, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

The humanized anti-A beta monoclonal antibody provided by the invention can inhibit A beta monomer polymerization, promote phagocytosis of A beta by macrophages, protect nerve cells from toxicity of A beta, and can be used for treating and diagnosing diseases and symptoms related to amyloidosis, such as Alzheimer's disease.

The invention also provides application of the humanized anti-A beta monoclonal antibody, the conjugate and/or the conjugate in preparation of a product for detecting A beta expression.

Experiments show that the humanized anti-A beta monoclonal antibody provided by the invention can be combined with an A beta monomer. Therefore, the humanized anti-A beta monoclonal antibody provided by the invention can be used for detecting an A beta monomer.

The invention also provides a kit comprising the humanized anti-a β monoclonal antibody, conjugates and/or conjugates thereof.

The kit for detecting the A beta monomer or polymer mixture further comprises a coating buffer solution, a washing solution, a sealing solution and/or a developing solution.

The coating buffer is a carbonate buffer.

The washing solution comprises PBS, Tween, sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

The blocking solution comprises PBS and BSA.

The color development liquid comprises a TMB solution, a substrate buffer solution and a stop solution.

The substrate buffer comprises citric acid and disodium hydrogen phosphate.

The stop solution is aqueous hydrogen peroxide solution.

The kit for detecting the cells with the surface expressing the A beta also comprises PBS, goat anti-mouse IgG Fc and a TITC secondary antibody.

The diagnostic method of a disease, detect the expression of A beta with the kit that the invention provides, judge whether to suffer from the disease according to the expression level of A beta; the diseases and disorders include amyloidosis, a group of diseases and disorders associated with amyloid protein, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as alzheimer's disease.

In some embodiments of the present invention, the criterion for determining whether a disease is present or absent according to the expression level of a β is: the detection sensitivity of the kit is less than 20pg/ml for normal people of 600-1000 pg/ml and AD patients of 200-450 pg/ml.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in molecular biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain. The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG1, IgG2, IgG3, IgG4), and mu heavy chains or other type equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) contain a variable region of about 110 amino acids at the NH 2-terminus and a kappa or lambda constant region at the COOH-terminus. The full-length immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) likewise comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

"humanized antibody" refers to an antibody that comprises CDR regions derived from a non-human antibody, and the remainder of the antibody molecule is derived from one (or more) human antibody. Furthermore, to preserve binding affinity, some residues of the backbone (referred to as FR) segment may be modified.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The medicament contains at least one functional component and also comprises a medicinal carrier. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.

As used herein, "CDR regions" or "CDRs" refer to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest,5th ed., u.s.department of Health and Human Services, NIH,1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

The invention provides an antibody humanization transformation method, which carries out reasonable antibody humanization design by FR transplantation on a reference multi-template to obtain a humanized antibody with the affinity equivalent to that of a murine antibody.

The preparation method of the humanized anti-A beta monoclonal antibody provided by the invention comprises the following steps:

step 1: preparing mouse hybridoma, and obtaining an antibody sequence through 5' RACE;

step 2: humanization of the antibody, sequence alignment on an NCBI tool, completion of humanization modification, and screening of the modified antibody.

Specifically, the method comprises the following steps:

the preparation method of the humanized anti-Abeta monoclonal antibody comprises the following steps: using a mouse antibody 066-5.4.1 as a template, performing PCR amplification to obtain an antibody heavy chain variable region gene VH and a light chain variable region gene VL, translating the genes into amino acid sequences, comparing the amino acid sequences with human antibody sequences in an NCBI database, respectively selecting 5 human antibody sequences with the highest similarity to the variable region VH and VL, using the sequences as a humanized transformation reference template, determining a CDR region of the mouse antibody 066-5.4.1, keeping the CDR region unchanged, transplanting FR regions in the 5 reference templates of the VH and VL respectively to 066-5.4.1 to obtain humanized sequences, and respectively constructing transient expression vectors after codon optimization; transferring the expression vector into 293E cells for expression to obtain a humanized antibody specifically combined with A beta; the humanized A beta antibody is finally obtained by affinity determination, determination of the value of EC50 bound with antigen, detection of the polymerization of A beta inhibition, detection of the activity of macrophage phagocytosis A beta and detection of the cytotoxic protection effect on the humanized antibody.

The humanized anti-A beta monoclonal antibody provided by the invention can inhibit A beta monomer polymerization, promote phagocytosis of A beta by macrophages, protect nerve cells from toxicity of A beta, and can be used for treating and diagnosing diseases and symptoms related to amyloidosis, such as Alzheimer's disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows SDS-PAGE and WB detection of A β monomers and polymer mixtures; lane M: protein molecular weight markers; lane 1: a β monomer; lane 2: a β polymer blend; fig. 1 (a): SDS-PAGE detection of A beta monomer and polymer mixture; FIG. 1 (B): WB detection of Abeta monomer and polymer mixture;

FIG. 2 shows SDS-PAGE electrophoresis to detect purified positive antibodies; lane M: protein molecular weight markers; lane 1: 066-P01 non-reducing electrophoresis; lane 2: 066-P01 reduction electrophoresis; lane 3: 066-P02 non-reducing electrophoresis; lane 4: 066-P02 reduction electrophoresis; wherein, fig. 2 (a): SDS-PAGE electrophoresis detection result of the purified positive antibody 066-P01; fig. 2 (B): SDS-PAGE electrophoresis detection result of the purified positive antibody 066-P02;

FIG. 3 shows that anti-A.beta.monoclonal antibodies inhibit A.beta.polymerization assays; the abscissa is different sample adding groups, the ordinate is relative fluorescence intensity, and the anti-Abeta monoclonal antibodies 066-4.22.1, 066-4.26.14, 066-5.4.1 and the like can inhibit Abeta polymerization; wherein, FIG. 3(A) shows the result of detecting the inhibition of Abeta polymerization by IgG, 066-P01, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1 and 066-6.7.2 anti-Abeta monoclonal antibodies; FIG. 3(B) shows the detection results of the inhibition of Abeta polymerization by the anti-Abeta monoclonal antibodies of PBS, IgG, 066-4.21.13, 066-4.26.14, 066-4.6.8, 066-4.22.1, 066-4.18.2 and 066-P01 in the sample-loading group;

FIG. 4 shows the measurement of macrophage phagocytosis of A.beta.by anti-A.beta.monoclonal antibody; the abscissa is different sample adding groups, the ordinate is fluorescence intensity, and the 066-5.4.1 and 066-7.17.2 anti-Abeta monoclonal antibodies have the activity of promoting macrophages to phagocytose Abeta;

FIG. 5 shows the anti-A β monoclonal antibody cytotoxicity protection activity assay; the abscissa is different sample adding groups, the ordinate is relative values of LDH release, and the 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2 and 066-7.17.2 antibodies have cytotoxic protection effects which are equivalent to 066-P02; wherein, FIG. 5(A) shows the detection results of cytotoxic protection activity of anti-Abeta monoclonal antibodies of Vehicle, IgG, 066-4.21.13, 066-4.17.28, 066-4.6.8, 066-4.22.1, 066-4.26.14, 066-4.18.2 and 066-P02 respectively; FIG. 5(B) shows the result of detecting the cytotoxic activity of anti-Abeta monoclonal antibodies of Vehicle, IgG, 066-6.2.1, 066-7.17.2, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.7.2, and 066-P02 for the loading groups, respectively;

FIG. 6 shows the results of the Morris water maze test; the abscissa is the time after treatment (days 1, 2, 3, 4, 5), and the ordinate is the time to find the hidden platform under the water (unit: Sec);

FIG. 7 shows total RNA agarose gel electrophoresis detection; lane M: DL2000 molecular weight markers; lane 1: 066-5.4.1 total RNA electrophoresis band;

FIG. 8 shows agarose gel electrophoresis detection of heavy chain variable regions and light chain variable regions of candidate antibodies amplified by PCR; lane M: DL2000 molecular weight markers; lane 1: 066-5.4.1 heavy chain variable region PCR product electrophoresis band; lane 2: 066-5.4.1 light chain variable region electrophoresis band; wherein, FIG. 8(A) shows the results of PCR for the heavy chain variable region from 066 to 5.4.1; FIG. 8(B) shows the results of PCR for the light chain variable region from 066-5.4.1;

FIG. 9 shows SDS-PAGE electrophoresis detection of purified murine human chimeric antibody 066-5.4.1-chb; lane M: protein molecular weight markers; lane 1: non-reduction electrophoresis of a mouse human chimeric antibody 066-5.4.1-chop; lane 2: reducing electrophoresis of a mouse human chimeric antibody 066-5.4.1-chop;

FIG. 10 shows SDS-PAGE electrophoresis detection of purified parent humanized candidate antibodies; lane M: protein molecular weight markers; in fig. 10 (a): lanes 1-6 are non-reducing electrophoresis with humanized antibodies 066-5.4.1H1L4, 066-5.4.1H2L5, 066-5.4.1H5L1, 066-5.4.1H5L2, 066-5.4.1H5L3, 066-5.4.1H5L4, respectively; in fig. 10 (B): lanes 1-6 are reductive electrophoreses with humanized antibodies 066-5.4.1H1L4, 066-5.4.1H2L5, 066-5.4.1H5L1, 066-5.4.1H5L2, 066-5.4.1H5L3, 066-5.4.1H5L4, respectively;

FIG. 11 shows a humanized A β antibody inhibition assay of A β polymerization; the abscissa is different sample adding groups, and the ordinate is relative fluorescence intensity, and the 066-5.4.1 humanized antibody can inhibit A beta polymerization;

FIG. 12 shows the assay of macrophage phagocytosis of A.beta.by humanized A.beta.antibody; the abscissa is different sample adding groups, the ordinate is fluorescence intensity, the 066-5.4.1 humanized candidate antibodies have the activity of promoting phagocytosis of Abeta by macrophages, and the 066-5.4.1H5L3 has the optimal performance;

FIG. 13 shows humanized A β antibody cytotoxic protection activity assay; the abscissa represents different loading groups, and the ordinate represents a relative value of LDH release, and the 066-5.4.1 humanized candidate antibodies all have cytotoxic protection effects which are equivalent to 066-P02.

Detailed Description

The invention discloses a humanized anti-Abeta monoclonal antibody and application thereof, and a person skilled in the art can realize the humanized anti-Abeta monoclonal antibody by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The humanized anti-Abeta monoclonal antibody and the raw materials and reagents used in the application thereof can be purchased from the market.

The invention is further illustrated by the following examples: